Multi-epitope vaccine against SARS-CoV-2 targeting the spike RBD: an immunoinformatics approach.

Aim: We designed a SARS-CoV-2 epitope vaccine based on the receptor-binding domain (RBD) in virus spike protein. Methods: RT-PCR performed on nasopharyngeal swab COVID-19 patients. After registering RBD region in the GenBank, physicochemical parameters, secondary structure, homology modeling, 3D structure of RBD region and antigenicity were determined using ProtParam ExPASy, PSIPRED, MolProbity, IEDB and Vaxijen online tools, respectively. Results: B and T cell epitopes were predicted in terms of non-allergenicity and antigenicity. MolProbity analysis provided a qualitative model for RBD. The homology model showed that most of the residues are in optimal district of energy. Conclusion: High immunogenicity score of epitopes indicates promising candidates for the development of multi-epitope vaccines. It may help to develop an effective vaccine.

In order to identify the sequence of RBD region of S protein in SARS-CoV-2, RT-PCR test was performed on nasopharyngeal swab samples of four COVID-19 patients referred to Imam Khomeini Hospital in Ardabil. After registering the sequence of the RBD region in the GenBank database, the physicochemical parameters, secondary structure, homology modeling, 3D structure of the RBD region and antigenicity were determined using ProtParam ExPASy, PSIPRED, MolProbity, IEDB and Vaxijen online tools, respectively.

Evaluation the reactivity of a peptide-based monoclonal antibody derived from OmpA with drug resistant pulsotypes of Acinetobacter baumannii as a potential therapeutic approach.

BACKGROUND: Acinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed a novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A. baumannii. METHODS: Peptide derived from A. baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into BALB/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, western blotting, indirect immunofluorescence (IFA), and in vitro opsonophagocytosis assays were used to evaluate the reactivity of generated mAb. RESULTS: The anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (1.94 × 10-9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased the bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement. CONCLUSIONS: The peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as a novel therapeutic approach.

Production and characterization of novel monoclonal antibodies against outer membrane protein A (OmpA) of Acinetobacter baumannii.

BACKGROUND: Infection caused by Acinetobacter baumannii has emerged as a significant clinical problem with unacceptably high mortality rate due to the increase in antibiotic-resistant strains. Producing novel monoclonal antibody (MAb) against outer membrane protein A (OmpA) could be considered as a potential tool to improve treatment of A. baumannii infections. OBJECTIVES: In this study, we aimed to produce murine MAbs against OmpA peptide of A. baumannii. MATERIALS AND METHODS: BALB/c mice were immunized with 18-mer amino acid peptide as a part of the OmpA protein. Four antibody-secreting hybridomas were obtained using hybridoma technology and then characterized according to isotypes, affinity constant, reactivity in ELISA, flow cytometry, indirect immunofluorescence (IFA) and opsonophagocytic killing assays. RESULTS: All four produced MAbs (1A1-D10, 1G1-E7, 2C11-F10, and 4H2-H9) had IgG1 isotype with Kappa light chain. One of these MAbs, 1G1-E7 was purified and selected for further characterizations. 1G1-E7 showed a high reactivity with both immunogenic peptide and A. baumannii in ELISA. Our results indicated that 1G1-E7 MAb reacted with 95.3% of A. baumannii in flow cytometry as well as IFA. Moreover, the affinity of the 1G1-E7 MAb was measured 1.37 × 108 M-1. The 1G1-E7 significantly improved opsonophagocytic killing of a clinical isolate of A. baumannii. CONCLUSION: Our findings showed that the OmpA can be identified by produced MAbs. The efficacy of novel anti-OmpA antibodies in A. baumannii targeting needs to be further investigated in challenging models, and then could be subjected for genetic engineering to produce therapeutic antibody against A. baumannii.

The Challenge of Global Emergence of Novel Colistin-Resistant Escherichia coli ST131.

Escherichia coli ST131 is one of the high-risk multidrug-resistant clones with a global distribution and the ability to persist and colonize in a variety of niches. Carbapenemase-producing E. coli ST131 strains with the ability to resist last-line antibiotics (i.e., colistin) have been recently considered a significant public health. Colistin is widely used in veterinary medicine and therefore, colistin-resistant bacteria can be transmitted from livestock to humans through food. There are several mechanisms of resistance to colistin, which include chromosomal mutations and plasmid-transmitted mcr genes. E. coli ST131 is a great model organism to investigate the emergence of superbugs. This microorganism has the ability to cause intestinal and extraintestinal infections, and its accurate identification as well as its antibiotic resistance patterns are vitally important for a successful treatment strategy. Therefore, further studies are required to understand the evolution of this resistant organism for drug design, controlling the evolution of other nascent emerging pathogens, and developing antibiotic stewardship programs. In this review, we will discuss the importance of E. coli ST131, the mechanisms of resistance to colistin as the last-resort antibiotic against resistant Gram-negative bacteria, reports from different regions regarding E. coli ST131 resistance to colistin, and the most recent therapeutic approaches against colistin-resistance bacteria.

Intra- and inter-examination repeatability of magnetic resonance spectroscopy, magnitude-based MRI, and complex-based MRI for estimation of hepatic proton density fat fraction in overweight and obese children and adults.

PURPOSE: Determine intra- and inter-examination repeatability of magnitude-based magnetic resonance imaging (MRI-M), complex-based magnetic resonance imaging (MRI-C), and magnetic resonance spectroscopy (MRS) at 3T for estimating hepatic proton density fat fraction (PDFF), and using MRS as a reference, confirm MRI-M and MRI-C accuracy. METHODS: Twenty-nine overweight and obese pediatric (n = 20) and adult (n = 9) subjects (23 male, 6 female) underwent three same-day 3T MR examinations. In each examination MRI-M, MRI-C, and single-voxel MRS were acquired three times. For each MRI acquisition, hepatic PDFF was estimated at the MRS voxel location. Intra- and inter-examination repeatability were assessed by computing standard deviations (SDs) and intra-class correlation coefficients (ICCs). Aggregate SD was computed for each method as the square root of the average of first repeat variances. MRI-M and MRI-C PDFF estimation accuracy was assessed using linear regression with MRS as a reference. RESULTS: For MRI-M, MRI-C, and MRS acquisitions, respectively, mean intra-examination SDs were 0.25%, 0.42%, and 0.49%; mean intra-examination ICCs were 0.999, 0.997, and 0.995; mean inter-examination SDs were 0.42%, 0.45%, and 0.46%; and inter-examination ICCs were 0.995, 0.992, and 0.990. Aggregate SD for each method was <0.9%. Using MRS as a reference, regression slope, intercept, average bias, and R (2), respectively, for MRI-M were 0.99%, 1.73%, 1.61%, and 0.986, and for MRI-C were 0.96%, 0.43%, 0.40%, and 0.991. CONCLUSION: MRI-M, MRI-C, and MRS showed high intra- and inter-examination hepatic PDFF estimation repeatability in overweight and obese subjects. Longitudinal hepatic PDFF change >1.8% (twice the maximum aggregate SD) may represent real change rather than measurement imprecision. Further research is needed to assess whether examinations performed on different days or with different MR technologists affect repeatability of MRS voxel placement and MRS-based PDFF measurements.

Production and Characterization of Monoclonal Antibodies against Human Prostate Specific Antigen.

BACKGROUND: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented. METHODS: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC). RESULTS: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/К) and clones (2C8-E9, 2G3-E2, IgG2a/К) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot. CONCLUSION: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

Immunohistochemical characterization of novel murine monoclonal antibodies against human placenta-specific 1.

Human PLAC1 (placenta-specific 1) is a new member of cancer-testis antigens with 212 amino acids, and its expression is restricted to placenta and at much lower levels to testis. Recently, ectopic expression of the PLAC1 transcript has been demonstrated in a wide range of human tumors and cancer cell lines with a proposed function in tumor cell growth. No monoclonal anti-PLAC1 antibody applicable to immunohis-tochemical staining is available so far. To better understand the PLAC1 expression and localization, we aimed to produce monoclonal antibodies (mAbs) against the extracellular region of PLAC1. Mice were immunized with a synthetic peptide corresponding to the C-terminal 11 amino acids of PLAC1 conjugated with a carrier protein. Hybridomas were produced by standard protocol and screened for positive reactivity by enzyme-linked immunosorbent assay. Reactivity of final two clones was then assessed by Western blotting (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC). Both clones showed a specific immunostaining pattern in human term placenta as the positive control. Reactivity was mostly localized to the cytoplasm of syncytiotrophoblasts. One of the clones showed an excellent staining signal in breast, ovary, and prostate cancer cell lines. Importantly, no reactivity was observed with human lymph node cells or prostate. None of the mAbs were able to detect PLAC1 in Western blot. Based on the present results, these mAbs can be used for detection of PLAC1 in IHC and ICC techniques.

Production and characterization of a murine monoclonal antibody against human ferritin.

BACKGROUND: Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. METHODS: Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. RESULTS: Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×10(9) M (-1)) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. CONCLUSION: This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met.

A survey on the prevalence of Chlamydia trachomatis and Mycoplasma genitalium infections in symptomatic and asymptomatic men referring to urology clinic of labbafinejad hospital, tehran, iran.

BACKGROUND: Chlamydia trachomatis and Mycoplasma genitalium infections are the most prevalent sexually transmitted bacterial infections in the world that cause urogenital infections in both men and women. It appears that infertility is a complication of these infections. OBJECTIVE: This study was designed to estimate the prevalence of Chlamydia trachomatis and Mycoplasma genitalium in symptomatic and asymptomatic men and to assess risk factors associated with infection. PATIENTS AND METHODS: Urine specimens were collected from 200 men; 100 of them were symptomatic and 100 asymptomatic. Samples were examined by PCR to detect the infections. RESULTS: C. trachomatis was detected in 20% of symptomatic and in 4% of asymptomatic men (P < 0.001). The prevalence of M. genitalium was revealed to be 12% and 2% in symptomatic and asymptomatic men, respectively (P < 0.01). Four of 100 men in the symptomatic group were infected with both organisms. C. trachomatis infection was associated with dysuria, urethral discharge, testicular swelling, and genital ulcer (P < 0.05). M. genitalium infection was related with dysuria, testis inflammation, pelvic pain and low educational level (P < 0.05). Furthermore, the prevalence of infections at ages 30-39 years was more than other ages. CONCLUSIONS: Considering the role of these bacteria in urogenital infections, a screening test is recommended. Since the PCR assay is a highly sensitive and specific assay for the detection of these bacteria in male urine specimens, it provides a noninvasive technique for routine screening.

Skull base aneurysmal bone cyst presented with foramen jugular syndrome and multi-osseous involvement.

Aneurysmal bone cyst (ABC) is an expansile bone lesion that usually involves the long bones. Skull base involvement is rare. Hereby, we describe a 17-year-old man with hoarseness, facial asymmetry, left sided sensorineural hearing loss and left jugular foramen syndrome. CT scan and MRI showed a skull base mass that was confirmed as ABC in histopathology. The case was unusual and interesting due to the clinical presentation of jugular foramen syndrome and radiological findings such as severe enhancement and multiosseous involvement.